Rho-dependent and Rho kinase-dependent interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) (A,B). Measurement of the interaction of (A) green fluorescent protein (GFP)-LIMK1 (A), or (B) GFP-LIMK2, with monomeric red fluorescent protein (mRFP)-fascin-1S39A in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for monomeric red fluorescent protein (mRFP) (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C), Percentage FRET efficiency under each experimental condition. Each column represents the mean from nine to sixteen cells per condition and three independent experiments; bars indicate SEM. *P < 0.01 versus control; **P < 0.005 versus control. (D) Representative immunoblots from pull-downs of SW480 cell lysates with hexahistidine (6His)-tagged fascin-1 (wild-type (WT), S39A or S39D) bound to nickel-agarose beads. (E) Quantification of LIMK1 binding to fascin-1 bead matrices. For each matrix, LIMK1 binding was ratioed to binding to the bead-only matrix, based on quantification of grayscale images in ImageJ software (http://rsb.info.nih.gov/ij/download.html). Each column represents mean values from three independent experiments; bars indicate SEM. (F) Demonstration that LIMK1 binding to 6His-fascin-1 depends on Rho and Rho kinase activities. Representative of three independent experiments. (D,F) Molecular markers are in kDa.